The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii
The GTPase Activity of Murine Guanylate-binding Protein 2 (mGBP2) Controls the Intracellular Localization and Recruitment to the Parasitophorous Vacuole of Toxoplasma gondii
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DOI：
10.1074/jbc.m112.379636
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发表时间：
2012-08-10
影响因子：
4.8
通讯作者：
Pfeffer, Klaus
中科院分区：
文献类型：
--
作者：
Kravets, Elisabeth;Degrandi, Daniel;Pfeffer, Klaus
不同细胞类型中最丰富的IFN-γ诱导蛋白家族之一是65-kDa鸟苷酸结合蛋白家族，其被募集到细胞内寄生虫弓形虫的寄生虫空泡中。在这里，我们阐明了mGBP 2响应弓形虫的生物化学与细胞宿主防御功能之间的关系。野生型蛋白质对鸟嘌呤核苷酸表现出低亲和力，在GTP结合后自组装，在活化状态下形成四聚体，并以协同方式刺激GTP酶活性。两个连续水解反应的产物都是GDP和GMP。mGBP 2的GTP结合基序中的点突变体的生物化学表征揭示了氨基酸残基，这些氨基酸残基使GTP酶活性降低了几个数量级，并强烈损害核苷酸结合和多聚化能力。活细胞成像采用多参数荧光图像光谱（MFIS）使用Homo-FRET测定表明，诱导mGBP 2的多聚化依赖于一个功能性的GT3结构域。一致的结果表明，GTP结合，自组装，和刺激的水解活性所需的蛋白质在感染和未感染的细胞中的生理定位。最终，我们表明，GTdR域调节T的有效招募。弓形虫对IFN-γ的反应。
One of the most abundantly IFN-gamma-induced protein families different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-gamma.